Annals of Botany 100: 1209–1217, 2007

doi:10.1093/aob/mcm217, available online at www.aob.oxfordjournals.org

The Endophytic System of Mediterranean Cytinus (Cytinaceae) Developing

on Five Host Cistaceae Species

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C L AR A DE V EG A* , P E DRO LU IS O RTI Z, MO NT SERR AT AR ISTA and S ALVAD OR TA LAVERA
Departamento de Biologı́a Vegetal y Ecologı́a, Facultad de Biologı́a, Universidad de Sevilla, Apdo-1095,
41080 Sevilla, Spain

Received: 28 May 2007 Returned for revision: 10 July 2007 Accepted: 20 July 2007 Published electronically: 5 September 2007

† Background and Aims One of the most extreme manifestations of parasitism is found in the genus Cytinus, a
holoparasite whose vegetative body is reduced to an endophytic system living within its host root. There are two
species of Cytinus in the Mediterranean, C. hypocistis and C. ruber, which parasitize various genera of
Cistaceae, one of the most characteristic families of the Mediterranean scrublands. The aim of this work is to
describe the endophytic systems of C. hypocistis and C. ruber, and their tissue relationships with their host.
† Methods Roots from five different hosts infected with C. hypocistis and C. ruber were harvested, and examined by
anatomical techniques under light microscopy to elucidate the characteristics of the endophytic system of Cytinus,
and to determine if differences in endophytic systems occur between the two species and in response to different
hosts.
† Key Results The endophyte structure is similar in both Cytinus species irrespective of the host species. In the initial
stages of the endophyte, rows of parenchymal cells spread through the host pericyclic derivatives and phloem, and
begin to generate small nodules in the outermost region of the host xylem. Later the nodules anastomose, and bands
of parasitic tissue are formed. The host cambium continues to develop xylem tissue, and consequently the endophyte
becomes enclosed within the xylem. The bands of parasitic tissue fuse to form a continuous sheath. This mature
endophyte has well-developed vascular system with xylem and phloem, and forms sinkers with transfer cells that
grow through the host xylem.
† Conclusions The endophytic system of Cytinus develops in all host root tissues and reaches its most mature stages
in the host xylem. It is more complex than previously reported, showing parenchyma, xylem and phloem tissues.
This is the first report of well-developed phloem in a holoparasitic endophytic species.

Key words: Cistaceae, Cytinaceae, Cytinus hypocistis, Cytinus ruber, endophyte, Mediterranean region, parasitic plant,
sieve elements, sinker, transfer cell.

IN TROD UCT IO N detailed data only for the genus Pilostyles (Dell et al.,
1982; Kuijt et al., 1985). The few existing data indicate
One of the most extreme manifestations of parasitism is
that these endophytes are composed of parenchymal cells,
found in the families of endoparasites Rafflesiaceae,
with xylem elements being observed only occasionally
Mitrastemonaceae, Apodanthaceae and Cytinaceae. These
(Fraysse, 1906; Kuijt, 1969; Dell et al., 1982; Forstmeier
perennial plants, without chlorophyll, are obligate parasites,
et al., 1983; Kuijt et al., 1985). Functional phloem has
and depend on their hosts to obtain water and nutrients
never been observed in the endophytes of these families
(Kuijt, 1969). All show a reduction in their morphological
studied to date, although Kuijt et al. (1985) have described
characters, with scale-like leaves and absence of external
a vestigial phloem tissue in Pilostyles thurberi
roots, and their vegetative body is reduced to a haustorial
(Apodanthaceae). Within the host, the endophytic system
or endophytic system, often compared with that of a
can grow intercellularly between the cambial zone and
fungal plectenchyme. These endophytes live within the
the phloem (Mitrastemonaceae), through the cambial zone
roots or stems of their hosts (Kuijt, 1969; Meijer, 1993),
(Rafflesiaceae, Apodanthaceae), through the phloem
and emerge from the hosts only during the reproductive
(Apodanthaceae), or between the cambial zone and the
period, when the inflorescences arise. Because of this
xylem (Cytinaceae) (Kuijt, 1969; Meijer and Schlauer,
characteristic lifestyle, these endophytic holoparasites
2002).
were long considered to constitute a single family, the
The distribution of the families of the holoparasitic endo-
Rafflesiaceae. However, differences in the morphology of
phytes is mainly tropical or subtropical (Molau, 1995), and
flowers, ovaries and seeds, together with data from recent
in the Mediterranean region there are only two species,
molecular phylogenetic studies, indicate that they are dis-
Cytinus hypocistis and Cytinus ruber (Cytinaceae) both of
tinct families, even belonging to different orders (Bouman
which parasitize exclusively roots of Cistaceae, one of the
and Meijer, 1994; Barkman et al., 2004; Nickrent et al.,
most characteristic families of shrubs in the Mediterranean
2004; Davis et al., 2007).
flora. Cytinus hypocistis has yellow flowers and parasi-
The structure of the haustorial system in these reduced
tizes white or yellow-flowered Cistus, Halimium,
endophytic holoparasites is poorly known, and there are
Helianthemum, while C. ruber has white or
pinkish-white flowers and parasitizes pink-flowered Cistus.
* For correspondence. E-mail cvega@us.es

# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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1210 de Vega et al. — Endophytic System of Mediterranean Cytinus Species

Although Mediterranean Cytinus have been known since specimens of the host plants were deposited in the herbar-
ancient times, and even mentioned by Teofrasto (Villar, ium of the University of Seville.
1997), their life cycle, floral biology, process of infection,
and relationhips with the hosts remain practically
unknown. The anatomy of the endophytic system of R E S U LT S
C. ruber is unknown and that of C. hypocistis poorly docu- Macroscopic morphology of the infection
mented, with almost all the existing studies going back a

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century or more, e.g. Solms-Laubach (1867); Fraysse Year after year, Cytinus inflorescences burst through the
(1906); and also Forstmeier et al. (1983). The aim of this same zone of the host root (observations made in 50 popu-
work is to describe the endophytic system of C. hypocistis lations over 5 years). Thus, a ‘reproductive zone’ of Cytinus
and C. ruber, and examine the relationships they establish on the host root can be referred to, that is generally found
with the host tissues. Moreover, given that the endophytes very close to the trunk. On the infected roots, both isolated
of some parasitic plants can adopt different forms depend- inflorescences of Cytinus and groups of up to 22 inflores-
ing on the host they parasitize (Thoday, 1960, 1963), the cences may occur on a relatively small portion of the root
development of the endophyte of Cytinus was studied on (Fig. 1A). Around the zone of emergence of each inflores-
five of its most common host species. This work falls cence a cup-shaped structure is formed that originates from
within a broad study on the Mediterranean Cytinus which host tissue that ruptures, enabling the inflorescence to exit
has shown that their taxonomic status is uncertain, since (Fig. 1B). These structures remain as permanent scars on
genetic races or cryptic species defined by host identity the roots.
seem to exist (C. de Vega, unpubl. res.). However, in the
absence of a new recognized classification, the two tra-
ditionally recognized species, C. hypocistis and C. ruber, Structure of the endophytic system and relationships
are dealt with here. with the host tissues
The anatomical structure of the roots of the five species
of Cistaceae studied is similar, showing the typical charac-
M AT E R I A L S A N D M E T H O D S teristics of dicotyledonous secondary roots. The outermost
layer of the root is a periderm, and internally a large zone
Cytinus hypocistis (L.) L. and C. ruber (Fourr.) Fritsch. are of pericyclic derivatives with abundant fibres and scarce
perennial holoparasites that appear exclusively on roots of intercellular spaces is found. The phloem does not show
Cistaceae species. The vegetative body of both parasites fibres or sclereids, and appears separated from the xylem
is contained entirely within the root of their host plants, by a narrow cambial zone. The xylem occupies the
and only in spring do the inflorescences burst through the
host tissues. According to Heinricher (1917) the develop-
mental time for Cytinus to produce an inflorescence
requires over 3 years.
Roots of Cistus albidus L. infected with Cytinus ruber,
and roots of Cistus ladanifer L., Cistus salviifolius L.,
Halimium halimifolium (L.) Willk. and Halimium
ocymoides (Lam.) Willk., infected with Cytinus hypocistis,
were collected from natural populations in south-west
Spain. Root collection was made during the flowering of
Cytinus, given that infestation is apparent only in this
period.
Small pieces of these roots were fixed in 4 % glutaralde-
hyde in 0.1 M phosphate buffer, pH 7.0, for approx. 1 d at
room temperature, and further incubated in a vacuum oven
for 1 h at room temperature to improve the fixation. The
tissues were subsequently dehydrated in an ethanol series,
embedded in Historesin (glycol methacrylate; Leica,
Heidelberg), and serially sectioned at 2 – 4 mm, using a
rotary microtome. Sections were stained with 0.12 %
toluidine blue and 0.05 % basic fuschin (Junqueira, 1990),
and mounted with Eukitt (Kindler, Freiburg, Germany).
For observation of sieve elements, sections were stained
with 0.05 % aniline blue in 0.05 M phosphate buffer ( pH
8.5) and then examined under a fluorescence microscope.
Under these conditions, sieve tubes were visible due to
fluorescent callose deposits. F I G . 1. (A) Inflorescences of Cytinus ruber emerging from a root of
All images were taken with a Leica DC300 camera Cistus albidus. (B) Detail of the scars left by previous inflorescences of
attached to a Zeiss Axiophot light microscope. Voucher Cytinus hypocistis on a root of Cistus salviifolius.
 de Vega et al. — Endophytic System of Mediterranean Cytinus Species 1211

greater part of the root, and the vascular elements are het-
erogeneous in size.
The development and structure of the endophytic system
of Cytinus are similar in both species studied in their five
host species. Thus, the following description is general,
and independent of the host being parasitized. The endo-
phytic system of Cytinus grows in all the tissues of the
region of the host xylem, contiguous with the cambial
zone (Fig. 2C). Initially, these nodules have only parench-
ymal cells recognizable by their dense cytoplasm, which
stains intensely (Fig. 2C). However, they soon thicken,
and appear as a differentiated central cambial zone sur-
rounded by parenchyma tissue (Fig. 2D). As the nodules
anastomose, bands of parasitic tissue are formed

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host roots, although it reaches the most mature and
complex stages in the host xylem. In the initial stages, par-
enchymal cells of the parasite are observed growing inter-
cellularly among the pericyclic and phloem cells of the
host. These parenchymal cells form single-rowed filaments
that spread tangentially through the pericyclic tissues and
phloem of the host, and radially towards the centre of the
root (Fig. 2A, B). The parenchymal cells of Cytinus are
easily distinguished from those of the host by their large
nuclei and thickened walls (Fig. 2B). When these parasitic
radial filaments cross the host cambial zone, they begin to
generate small nodules of parasitic tissue in the outermost
(Fig. 2D). These bands show a clear differentiation of two
layers of parenchymal tissue enveloping a central cambial
zone (Fig. 2D). As the host cambium continues its activity,
producing xylem inwardly, the endophyte bands at this
stage are separated from the host cambial zone by its
xylem, but connections are maintained with the radial fila-
ments from which the nodules originated (Fig. 2D). As the
endophyte bands separate from the host cambial zone, the
parasite cambium begins to form vascular tissues, with
phloem externally and xylem internally, and the number
of cell strata in the parenchymal layers increases
(Fig. 2E). The sieve elements of the endophyte are larger

F I G . 2. Different stages in the development of the endophytic system of Cytinus hypocistis and C. ruber. (A) Cross-section of a root of Cistus salviifolius.
Arrows indicate the parasitic tissue. (B) Magnification of the area marked in red in (A), with the tissue of C. hypocistis coloured pink. Note the parasitic
cells crossing the host pericyclic tissues and phloem. These cells are easily distinguished from those of the host by their great size and large nucleus.
(C) Cross-section of a root of C. salviifolius with nodules of the endophyte in different stages of development. In the nodule on the right there are intensely
stained parenchymal cells (arrowhead) and a cambial zone (arrow). (D) Cross-section of a root of C. salviifolius, showing an endophytic band embedded
in the host xylem, formed by a cambial zone bounded externally and internally by layers of parenchyma (arrows). Arrowheads indicate radial rows of
endophyte cells. (E) Cross-section of a root of C. albidus, showing a mature stage of the endophyte in the host xylem. In the central zone of the endophyte
a cambial zone can be seen, and phloem and xylem vascular tissues. The arrows indicate sinkers. Abbreviations: HPe, host pericyclic tissues; HCa,
Host cambial zone; HP, host phloem; HX, host xylem; PC, parasite cambial zone; PP, parasite phloem; PX, parasite xylem. Scale bars: A ¼ 200 mm;
B–D ¼ 50 mm; E ¼ 100 mm.
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de Vega et al. — Endophytic System of Mediterranean Cytinus Species
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 de Vega et al. — Endophytic System of Mediterranean Cytinus Species 1213

than those of the host, and are more scattered (Fig. 3A, B).
As observed under fluorescence, no direct connections were
seen between the phloem of the endophyte and that of the
host. The sieve elements of the endophyte are long, with
thin walls, and a weakly staining cytoplasm lacking a
nucleus. The simple sieve plates found in the cross-walls
of Cytinus sieve elements are easily observed with fluor-
In the flowering period of Cytinus, the mature endophyte
grows towards the outside of the host root, causing a
massive distortion in its tissues, to form the inflorescence
(Fig. 4J). From the continuous sheath of the endophyte,
the bud from which the inflorescence originates begins to
develop, formed by elongated parenchymal cells with
very large nuclei (Fig. 4K, L) and by vascular filaments

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escence microscopy, after staining with aniline blue, as
are its lateral sieve areas (Fig. 3C, D). The elements of
the endophytic xylem vessels have walls with reticu-
lated or helicoidal secondary thickening (Fig. 3E and F,
respectively).
As the cambium of the host deposits new layers of xylem
on the parasitic tissue, the endophytic tissue becomes pro-
gressively more deeply embedded in the host xylem the
older the endophyte. At this stage, nodules formed under
the host cambial zone begin to form new endophyte
bands (Fig. 3G). The repetition of this process gives raise
to alternate strata of parasitic and host tissues (Fig. 3H).
As the bands of endophyte become encapsulated within
the host xylem, they fuse to form a continuous sheath
(Fig. 3I).
Even before the endophyte forms this sheath, radial pro-
cesses, called sinkers (Kuijt, 1977), also begin to differen-
tiate from the parasite cambial zone. The sinkers increased
in length with growth from the endophyte cambial zone,
and they seem to develop in conjunction with xylem develop-
ment. A sinker is generally single-rowed (Fig. 4A) and
extends through intercellular spaces until it encounters
mature host xylem where its terminal cell wedges itself
between xylem cells given rise to a pointed end (Fig. 4B,
C). The sinkers are generally formed of thick-walled par-
enchymal cells and where these cells are contiguous with
host vessels the wall is thickest against the vessel
(Fig. 4D). These parenchyma cells may have differentially
thickened walls that occur in contact with host vessels
(Fig. 4E), and so they can be considered as transfer cells,
particularly since such thickening of the sinker walls is
absent when they are abutting host fibres and parenchyma.
Occasionally isolated xylem cells of the parasite are inter-
calated between sinker parenchymal cells, but they are
easily recognizable by their helicoidal thickenings and by
their radial growth in the root (Fig. 4F). In no case were
phloem elements detected in the sinkers. Sometimes a
sinker seems to invade the elements of the host xylem
vessels, penetrating into their lumen and crossing several
successively (Fig. 4G – I).
arranged cylindrically close to the periphery (Fig. 4L– N).
Each vascular filament consists of xylem on its inner face
and phloem on the outer (Fig. 4M). The phloem tissue of
the inflorescences includes sieve elements similar to those
described above, and associated parenchymatic cells
(Fig. 4M, N). The latter are easily distinguishable because
of their intensely stained protoplasts, their large nuclei,
and their position beside the sieve elements.
DISCUSSION
The endophytic systems of Cytinus ruber and C. hypocistis
are alike, and develop similarly irrespective of the host
parasitized. The endophytic system of these species pro-
duces lateral filaments that extend tangentially through the
pericyclic derivatives and phloem of the root host. At
the same time, this endophyte grows radially, crossing the
cambial zone of the host and colonizing its xylem region.
Thus, the endophytic system of both species of Cytinus
grows in all the host root tissues. Most endophytic holo-
parasites develop only in the host cortex and the outermost
zone of the phloem (Brown, 1912; Kuijt, 1969; Rutherford,
1970; Kuijt et al., 1985), and only in Pilostyles hamiltonii
(Apodanthaceae) do some cellular filaments of the endo-
phyte penetrate into the xylem (Dell et al., 1982). In con-
trast, the endophyte of Cytinus reaches its highest degree
of complexity in the root xylem of its hosts. This type of
development has not been reported previously in any
species of holoparasitic endophyte. The fact that nodules
of Cytinus develop in the cambial zone and grow non-
intrusively into the xylem raises the possibility of a
developmentally synchronized growth of Cytinus and its
host, as has been reported in other parasites (Srivastava
and Esau, 1961; Lye, 2006).
One of the most important findings of the present study is
the detection of well-developed phloem in the endophyte of
Cytinus, with companion cells and sieve elements similar to
those of the autotrophic angiosperms. Previously it had
been reported that the endophyte of C. hypocistis comprised

F I G . 3. (A–F) Vascular tissues of the endophytic system of Cytinus hypocistis and C. ruber. (A) Longitudinal section of a root of Cistus ladanifer
infected by C. hypocistis, under normal light. (B) The same section, under fluorescence, showing the phloem of C. hypocistis (white arrows), oriented
perpendicular to that of the host (black arrows). (C) Detail of sieve tubes of (B), showing the sieve plates of C. hypocistis. (D) Detail of sieve elements
of C. hypocistis on Halimium halimifolium, showing the sieve plates and lateral sieve areas (arrows). (E) Detail of the xylem vessels of C. ruber on Cistus
albidus, showing the secondary reticulated thickening of the cell wall. (F) Detail of the xylem vessels of C. ruber on Cistus albidus, showing the sec-
ondary helicoidal thickening of the cell wall. (G– I) Mature stages in development of C. hypocistis. (G) Cross-section of a root of Halimium ocymoides,
showing the formation of new endophytic nodules (arrows) and, below, a band of older parasite tissue (arrowhead). (H) Cross-section of a root of
C. salviifolius with various strata of the endophytic system of C. hypocistis alternating with host xylem. The arrows indicate the radial rows of endophyte
cells. (I) Very advanced stage of the infection of C. hypocistis observed in a cross-section of a root of C. salviifolius: the endophyte forms a continuous
sheath embedded in the host xylem. The arrows indicate the radial rows of endophyte cells and the arrowheads the sinkers in the host xylem.
Abbreviations: HPe, host pericyclic tissues; HP, host phloem; HX, host xylem; PPa, parasite parenchyma. Scale bars: A, B, G ¼ 100 mm; C, E, F ¼
10 mm; D ¼ 5 mm; H, I ¼ 200 mm.
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only a cortical outer layer, a cambial zone, and a medullary
inner layer with xylem conductive elements (Solms-
Laubach, 1867; Fraysse, 1906; Forstmeier et al., 1983).
This was probably because fluorescence was not used in
those studies and, despite the size of the phloem elements,
they are barely perceptible without this technique (see
Fig. 3A and B). Likewise, phloem has not been reported
mature endophyte bands of Cytinus and the host xylem.
This seems to be a common circumstance, because although
xylem – xylem contacts have been reported in other parasitic
angiosperms (e.g. Calvin, 1967; Kuijt, 1977; Sallé, 1979;
Heide-Jørgensen and Kuijt, 1995; Pate, 1995; Calladine
and Pate, 2000), a luminal continuity was actually seen in
very few cases (e.g. Pate et al., 1990; Dörr, 1997).

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previously in any of the studies carried out on species
of Hydnoraceae, Mistratemonaceae or Rafflesiaceae.
Sieve elements were detected in Pilostyles turberi
(Apodanthaceae; Kuijt et al., 1985), but they were classed
as a vestigial tissue. This study of Cytinus species is the
first time that the presence of well-developed phloem has
been reported in a holoparasitic endophyte species.
Moreover, reports of sieve elements in holoparasitic
species whose vegetative bodies mainly develop outside
the host are few, e.g. Orobanche (Dörr and Kollmann,
1975, 1995), Epifagus (Walsh and Popovich, 1977) and
Aeginetia (Rajanna et al., 2005), although the existence
of phloem in hemiparasitic angiosperms has been documen-
ted on many occasions (for reviews, see Kuijt, 1977;
Bhandari and Mukerji, 1993). Direct contact was not
detected between the phloem of Cytinus and that of the
host, a type of contact infrequent in other parasites (Tate,
1925; Dörr, 1972; Dörr and Kollmann, 1995; Birschwilks
et al., 2006).
The endophyte of Cytinus produces sinkers that
thoroughly permeated the host xylem, parallel to the
xylem rays but not associated with them. In contrast, the
sinkers of many hemi- and holoparasites grow closely
associated with the host medullary rays (e.g. Cohen,
1954; Srivastava and Esau, 1961, Rutherford, 1970). It
has been reported that the sinkers of C. hypocistis were
comprised solely of parenchymal tissue (Fraysse, 1906;
Forstmeier et al., 1983), as in other endophytic holoparasite
species (Rutherford, 1970; Dell et al., 1982; Kuijt et al.,
1985). In this work, it has been observed that the sinkers
of both species of Cytinus also have tracheal elements
like those seen in hemiparasitic angiosperms (e.g.
Srivastava and Esau, 1961; Sallé, 1979). Despite the tra-
cheal elements detected in the sinkers of Cytinus, no
direct parasite xylem– host xylem contact was observed.
Nor were contacts observed between the xylem of the
Thus, in Cytinus, direct xylem – xylem or phloem –
phloem contacts have not been observed. It has been pro-
posed that the transfer of nutrients between parasitic
plants and hosts involves the parenchymal cell walls of
the haustorium, which could act as transfer cells, forming
an apoplastic continuum between the cells of the parasite
and those of the host (Coetzee and Fineran, 1987; Kuo
et al., 1989; Gedalovich-Shedletzky and Kuijt, 1990;
Heide-Jørgensen and Kuijt, 1993; Pate, 1995). Transfer
cells occur in most angiosperms, both parasitic and non-
parasitic, and they have a central role in nutrient distri-
bution, facilitating the apo-symplastic transport of solutes
(Fineran and Calvin, 2000; Christensen et al., 2003;
Offler et al., 2003). Contacts between the endophyte of
Cytinus and its host also seem to occur via parenchymal
cells. The parenchyma cells of the bands and sinkers with
thickened walls may act as transfer cells and, if so, they
would play a crucial physiological role in the host– parasite
interface, facilitating the absorption, transport and distri-
bution of photoassimilates from host to parasite. The devel-
opment of differentially thickened walls in the parenchyma
cells of the sinkers contiguous with vessels of the host
xylem suggests an active loading of water and solutes by
Cytinus from its host. Furthermore, it is possible that the
cells of the endophyte of Cytinus situated among the host
phloem cells capture photoassimilates from them.
Subsequently, the metabolites acquired could be trans-
ported, either symplastically or apoplastically, from the
portion of the endophyte situated in the pericyclic tissues
and phloem of the host, via sinkers, to the bands of
mature endophytic tissue embedded in the host xylem.
Thus, although the host tends to enclose Cytinus in its
xylem, the parasite may achieve its nutrient requirements
because of these connections.
Occasionally sinkers of Cytinus appear to penetrate the
host xylem cells. An intrusion of the sinkers into the host

F I G . 4. (A–H) Structure and development of the sinkers of Cytinus hypocistis and C. ruber. (A) Detail of C. ruber on Cistus albidus in a cross-section,
showing sinkers (arrows) being formed from the cambial zone of endophyte bands growing among the host xylem. (B) Sinkers of C. hypocistis invading
the xylem of Halimium halimifolium, in a cross-section. (C) Detail of sinkers of C. hypocistis, with thick cell walls and pointed apical cells (arrows), in the
xylem of Halimium ocymoides. (D, E) Sinkers of C. hypocistis on H. halimifolium. Note the presence of thick accumulations of wall material (D) and
differently thickened walls (E) in sinkers cells abutting host vessel elements (arrows). (F) Detail of a uniseriate sinker of C. hypocistis in the xylem of
H. ocymoides, showing a vascular element (arrow) between two parenchymal cells (arrowheads). (G) Longitudinal section of the xylem of H. halimifolium
thoroughly permeated by sinkers of C. hypocistis with cells having a large nucleus. (H) Longitudinal section of the xylem of H. halimifolium, showing a
sinker of C. hypocistis (arrow) that seems to invade two vascular elements of the host. (I) Longitudinal section of the xylem of H. halimifolium with
sinkers of C. hypocistis (arrows) that seems to penetrate the host vessels. (J–N) Details of the endophyte of Cytinus hypocistis and C. ruber forming
flower buds. (J) Cross-section of a root of Cistus albidus, showing the growth of the endophyte to form the inflorescence. (K) Detail of the large
nuclei of the endophyte cells from which the floral buds of C. hypocistis originate, in a longitudinal section of a root of C. salviifolius. (L)
Magnification of the marked area in (J) showing a parenchyma composed of long cells and vascular tissues (arrow). (M) Magnification of the marked
area in (L) showing xylem and phloem. In the xylem, the helicoidal thickenings of the walls can be observed, and in the phloem, the sieve elements
(arrows) and their associated parenchyma are evident. (N) Detail of the phloem, under fluorescence, of a floral bud of C. hypocistis on Cistus ladanifer,
showing sieve cells with a large amount of callose on their sieve plates. Abbreviations: HPe, host pericyclic tissues; HP, host phloem; HX, host xylem; PE,
endophytic system of Cytinus; PI, parasite inflorescence; PP, parasite phloem; PPa, parasite parenchyma; PX, parasite xylem. Scale bars: A, B, G, H, I, L,
N ¼ 50 mm; C, E, F, K, M ¼ 20 mm; D ¼ 10 mm; J ¼ 500 mm.
1216 de Vega et al. — Endophytic System of Mediterranean Cytinus Species
xylem vessels has also been reported in other parasitic
angiosperms (Kuijt, 1977; Dörr, 1997; Calladine and Pate,
2000), and it has been postulated that this penetration
might be produced by both mechanical pressure and enzy-
matic activity (Press and Whittaker, 1993; Dörr, 1997).
Such intrusive sinkers of Cytinus could take water and
mineral nutrients from the host, although this uptake
would be prevented if host vessels were occluded by
tyloses. Tylosis formation is considered a normal phenom-
enon in many species but it can also be induced by mech-
anical injury and by diseases (Fahn, 1977). In fact, Fraysse
(1906) found tyloses in roots of Cistaceae infected with
C. hypocistis. However, tyloses were not observed in
vessels contiguous with sinkers cells in any of the material
used in the present study.
Finally, for the first time, continuity has been demon-
strated here between the vascular tissues (xylem and
phloem) of the endophyte of Cytinus and those of its inflor-
escences, ensuring the flow of water and nutrients necessary
for flowering and fruiting. Although holoparasitic angios-
perms lack chlorophyll, and need to obtain water and nutri-
ents from their hosts, they sometimes show a small capacity
to assimilate carbon independently of the host (Renaudin
et al., 1982, 1984; Rey et al., 1985; Stewart and Press,
1990). Thalouarn et al. (1986), using radioisotope tech-
niques, have demonstrated that inflorescences of
C. hypocistis detached from the host are capable of non-
photosynthetic carboxylation using external CO2 in small
amounts – up to 12 % of the carbon received from the
host. Thus, C. hypocistis may obtain from its inflorescences,
extra nutrients that qualitatively complement those supplied
by the host, and which can be easily transported thanks to
the continuity of the conductive elements described in
this work.
CO N CLU DI NG R E MAR KS
The endophytic system described for both species of
Cytinus is more complex and extends deeper into the host
tissues than previously reported. The endophytic system
grows in all host root tissues, the youngest tissues being
located in the host pericyclic tissues and phloem and the
oldest tissues in the host xylem, with connections being
established between the different developmental stages of
parasitic tissues by means of sinkers. The mature endophy-
tic system has a well-developed vascular system with xylem
and functional phloem. The latter, as observed in Cytinus, is
described for the first time in any member of endophytic,
holoparasitic parasite. Sinkers formed from the mature
endophyte grow through the host xylem, and their cells
with thickened walls may act as transfer cells. Although
the current study presents a number of very novel features
of the endophytic system of Cytinus, little is known still
about its relationship with the host species from the first
stages of development. Since seed germination has never
been observed in any of the families of endophytic holopar-
asitic plants, the initial establishment and development of
Cytinus on the roots of its host species remains an open
and exciting question.
ACK N OW L E D G E M E N T S
We thank Dr R. G. Albaladejo for help with the field
sampling, Dr D. G. Simão and Dr R. Carmo-Oliveira for
technical assistance and Dr P. E. Gibbs for checking the
English text. This study was carried out in the Laboratory
of Morphology, Microscopy and Image Processing of the
Downloaded from https://academic.oup.com/aob/article-abstract/100/6/1209/126707 by Universitat de Barcelona. CRAI user on 01 April 2020
Institute of Biology-UFU, MG, Brazil. This work was sup-
ported by a PhD grant from the Spanish MEC to C. de Vega
and by two projects from the Spanish CICYT
(REN2002-04354-C02-02 and CGL 2005-01951).
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